Synthesis and Cleavage of Oligodeoxynucleotides Containing a 5-Hydroxyuracil Residue at a Defined Site

Abstract

Oxidation and hydrolysis of a cytosine residue can lead to the formation of 5-hydroxyuracil in DNA. The biological consequences of this modification are not fully understood. To facilitate biochemical and biophysical studies aimed at elucidating the effects of this modification in DNA, we have developed a solid-phase synthetic method for the placement of 5-hydroxyuracil residues at defined sites in oligodeoxynucleotides. This method is based upon the enhanced acidity of the 5-hydroxyl proton which allows selective aqueous acetylation. Under standard aqueous ammonia deprotection conditions, however, we observed that 5-hydroxyuracil residues are lost substantially from synthetic oligonucleotides. Substitution of aqueous ammonia with methanolic potassium carbonate and the use of phosphoramidite derivatives with alternatively protected amino groups allow synthesis of oligonucleotides containing 5-hydroxyuracil and all normal bases in high yield. The composition of the oligodeoxynucleotides prepared by this method has been verified by enzymatic digestion followed by high-performance liquid chromatography (HPLC) analysis as well as acid hydrolysis followed by GC/MS analysis. The location of the 5-hydroxyuracil residue is demonstrated by selective permanganate oxidation of the 5-hydroxyuracil residue followed by beta-elimination. We have also probed a synthetic oligonucleotide containing a unique 5-hydroxyuracil residue with uracil DNA N-glycosylase, previously reported to remove this lesion from DNA.

Mechanisms of Base Selection by Human Single-stranded Selective Monofunctional Uracil-DNA Glycosylase

“…Oligonucleotides were prepared by solid phase synthesis methods as described previously (25)(26)(27)(28). Following synthesis and deprotection, oligonucleotides were purified with Poly-Pak II cartridges and were denaturing gel-purified when necessary.…”

Synthesis and base pairing properties of DNA-RNA heteroduplex containing 5-hydroxyuridine

“…As shown in scheme 1 (A), compound 2 was prepared via bromination followed by hydrolysis in water from compound 1 which was commercially available (Tokyo Chemical Industry Co. Japan) (27). After protecting the two hydroxyl groups at 2'-and 3'-positions using isopropylation in acetone containing 2,2-dimethoxypropane, the hydroxyl group at the 5-position was acetylated in an aqueous medium to produce compound 3 (28).…”

Characterization of synthetic oligonucleotides containing biologically important modified bases by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

“…Oligonucleotides were detritylated using 80% aqueous acetic acid at room temperature for 30 min and then purified using a Waters XTerra C-18 column and a gradient of 0 to 20% acetonitrile in water (44). The base composition of the oligonucleotides reported here was verified by formic acid hydrolysis followed by GC/MS analysis (7,10,14,16,21).…”

“…Modified bases can occur in DNA as a consequence of chemical damage as well as incorporation of base analogs, many of which are important drugs (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20). Synthetic oligonucleotides containing modified bases are commonly used to determine the biological consequences of base substitutions and DNA damage.…”

“…Methods for the preparation of oligonucleotides containing phosphoramidites for 5-chlorouracil (40), 5-formyluracil (41), 5-hydroxyuracil (14,15), 5-carboxyuracil (17), and 5-(hydroxymethyl)uracil (42) have been described previously. Standard cyanoethyl phosphoramidites were used when possible and these oligonucleotides were deprotected with concentrated aqueous ammonia (28 to 30% as NH 3 ) at 60°C for 12 hours.…”